Protein folding is a problem of great significance in biochemistry. The ability to predict protein structure and function from the primary sequence of a protein would be of great importance to the development of protein-based pharmaceuticals. Similar, knowledge of the actual mechanism by which proteins fold, could lead to a better understanding of molecular diseases and allow the design of protein pharmaceuticals which avoid folding traps. Much has been learned about how interactions in the native state of a protein stabilize its three-dimensional structure. However, much less is understood about the other half of the protein folding equilibrium, the denatured state. NMR studies have shed light on some of the structural properties of this state. However, little is known about the relationship between structural changes and free energy in this loosely defined state. This laboratory has recently developed a means of assessing mutation-induced denatured stated free energy changes. The method involves measurement of in the bond strength of histidine-heme ligation in denatured iso-1-cytochrome c. In this proposal, this technique will be used to: evaluate deviations in random coil behavior for denture iso-1- cytochromes c with histidine at different positions in t he sequence with respect to the heme. evaluate the consequences of second site variants both near to and far from the histidine responsible for histidine-heme ligation in denatured iso-1-cytochrome c. use small heme-peptides to evaluate local versus long-range effects on denatured state stability. assess the dependence of denatured state free energy on denaturant concentration. This set of experiments will provide much needed knowledge about the energy landscapes of denatured proteins, which will be of great importance in defining the protein folding.